Mast cells are able to respond to a wide variety of stimuli such as antigen, substance P, and C3a. IgE, produced by allergic individuals binds to the high affinity IgE receptor (FcepsilonRI) found on mast cells reactivated through FcepsilonRI. Activation of tyrosine kinases in turn leads to actin polymerization and more rearrangement of the cytoskeleton. Thus, there is an active interplay between actin microfilaments and tyrosine kinases. The overall purpose of th project is to investigate the relationship between tyrosine kinases and the cytoskeleton and to determine which kinases are regulating actin polymerization and which kinases are being regulated by microfilaments. The first specific aim is to determine the role of adhesion and spreading in the activation of tyrosine kinases through Fcepsilon RI. This will be accomplished by monitoring the activity of several tyrosine kinases as well as their association with the cytoskeleton in non-adherent versus spread cells activated through FcepsilonRI. Mast cells which are lacking individual tyrosine kinases or which express dominant negative mutants of these kinases will also be used. In the second aim, the association of the tyrosine kinase, Btk, with microfilaments through it's PH domain will be explored. This tyrosine kinase is also believed to be involved in both antigen induced degranulation and actin polymerization. This will be done using transfected mast cells expressing dominant negative mutations of the PH domain. Biochemical studies as well as confocal immunofluorescent microscopy will be used in co- localization experiments. The purpose of the final aim is to investigate the chemotactic response of mast cells to C3a. Thus also involves tyrosine kinase driven actin polymerization. Cross-talk between the C3a receptor signaling pathways and FcepsilonRI signaling pathways will be explored.